Although much is known about the neural systems and neurotransmitters that regulate sleep and wakefulness, studies of the molecular regulation of sleep/wake states are only beginning to identify key molecules in intracellular signaling pathways that control these processes. The cyclic AMP-response element binding protein (CREB) is an activity-dependent transcription factor important for synaptic plasticity and memory storage. In rodents, levels of phosphorylated CREB within the cortex are higher in waking than in sleep, suggesting that CREB may play a role in sleep/wake regulation in mammals. Our studies of sleep/wake states in mice lacking the alpha and delta isoforms of Creb demonstrate that CREB acts to promote wakefulness. Over a 24 period, wake was significantly decreased in Creb alpha/delta mutant mice by approximately 100 minutes, and time spent in non-rapid eye movement (NREM) sleep was correspondingly increased. This decrease in wakefulness was largely due to an inability of Creb mutant mice to maintain the longer periods of wakefulness that occur during the nocturnal active period. Creb alpha/delta mice had decreased levels of theta activity during wake and REM sleep, suggesting that CREB plays a critical role in cortical arousal. Our data, together with that from Drosophila, indicate that there are, also, molecular mechanisms that promote wakefulness, possibly driven by wake-active neurotransmitters, such as norepinephrine. Indeed, norepinephrine can function to activate CREB. We hypothesize that CREB activity is normally altered by state-related changes in neural function; in turn, a suite of genes is activated by CREB and these genes subserve the functions of waking. We further hypothesize that a group of CREB-dependent genes will be induced with a slower time course and these will promote sleep. In this project, we outline experiments (Specific Aim 1) to examine the activity of CREB during extended wakefulness and across the diurnal cycle in neural circuits involved in sleep/wake regulation using immunochemical and Western blot experiments. In Specific Aim 2, we will identify targets of CREB important for sleep/wake regulation by analyzing knockout mice lacking the CREB target gene zif268 and by carrying out gene expression studies. In Specific Aim 3, we will use genetically modified mice lacking the gene encoding dopamine beta-hydroxylase, an enzyme required for the biosynthesis of norepinephrine and epinephrine, to determine if adrenergic signaling acts via CREB to maintain wakefulness. These proposed experiments will help define the molecular mechanisms by which wakefulness is maintained and sleep is promoted.